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  Method for producing l-amino acids by fermentation using bacteria having enhanced expression of xylose utilization genesUSPTO Application #: 20050214911Title: Method for producing l-amino acids by fermentation using bacteria having enhanced expression of xylose utilization genes Abstract: A method for producing an L-amino acid, such as L-histidine, L-threonine, L-lysine, L-glutamic acid, and L-tryptophan, using bacterium belonging to the genus Escherichia which has increased expression of genes, such as those of the xylABFGHR locus, which encode the xylose utilization enzymes, is disclosed. The method includes cultivating the L-amino acid producing bacterium in a culture medium containing xylose, and collecting the L-amino acid from the culture medium. (end of abstract)
Agent: Cermak & Kenealy LLP Acs LLC - Alexandria, VA, US Inventors: Aleksey Nikolaevich Marchenko, Sergey Vladimirovich Benevolensky, Elena Vitalievna Klyachko, Yuri Ivanovich Kozlov, Elvira Borisovna Voroshilova, Mikhail Markovich Gusyatiner USPTO Applicaton #: 20050214911 - Class: 435106000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Preparing Alpha Or Beta Amino Acid Or Substituted Amino Acid Or Salts Thereof The Patent Description & Claims data below is from USPTO Patent Application 20050214911. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application claims the benefit of U.S. provisional patent application No. 60/610,545 filed on Sep. 17, 2004, under 35 USC .sctn.119(e) and U.S. patent application Ser. No. 11/059,686 filed on Feb. 17, 2005 as a continuation-in-part under 35 U.S.C. .sctn.120. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The present invention relates to a method for producing L-amino acids by pentose fermentation, and more specifically to a method for producing L-amino acids using bacteria having enhanced expression of xylose utilization genes by fermentation of mixture of arabinose and/or xylose along with glucose as a carbon source. The non-expensive carbon source which includes a mixture of hexoses and pentoses of hemicellulose fractions from cellulosic biomass can be utilized for commercial production of L-amino acids, for example, L-histidine, L-threonine, L-lysine, L-glutamic acid, and L-tryptophan. [0004] 2. Brief Description of the Related Art [0005] Conventionally, L-amino acids have been industrially produced by fermentation processes using strains of different microorganisms. The fermentation media for the process typically contains sufficient amounts of different sources of carbon and nitrogen. [0006] Traditionally, various carbohydrates such as hexoses, pentoses, trioses; various organic acids and alcohols are used as a carbon source. Hexoses include glucose, fructose, mannose, sorbose, galactose and the like. Pentoses include arabinose, xylose, ribose and the like. However, the above-mentioned carbohydrates and other traditional carbon sources, such as molasses, corn, sugarcane, starch, its hydrolysate, etc., currently used in industry are rather expensive. Therefore, finding alternative less expensive sources for production of L-amino acids is desirable. [0007] Cellulosic biomass is a favorable feedstock for L-amino acid production because it is both readily available and less expensive than carbohydrates, corn, sugarcane or other sources of carbon. Typical amounts of cellulose, hemicellulose and lignin in biomass are approximately 40-60% of cellulose, 20-40% of hemicellulose 10-25% of lignin and 10% of other components. The cellulose fraction consists of polymers of a hexose sugar, typically glucose. The hemicellulose fraction is made up of mostly pentose sugars, including xylose and arabinose. The composition of various biomass feedstocks is shown in Table 1 (http://www.ott.doe.gov/biofuels/understanding_biomass.html). 1TABLE 1 Six-carbon Five-carbon Material sugars sugars Lignin Ash Hardwoods 39-50% 18-28% 15-28% 0.3-1.0% Softwoods 41-57% 8-12% 24-27% 0.1-0.4% [0008] More detailed information about composition of over 150 biomass samples is summarized in the "Biomass Feedstock Composition and Property Database" (http://www.ott.doe.gov/biofuels/progs/search1.cgi). [0009] An industrial process for effective conversion of cellulosic biomass into usable fermentation feedstock, typically a mixture of carbohydrates, is expected to be developed in the near future. Therefore, utilization of renewable energy sources such as cellulose and hemicellulose for production of useful compounds is expected to increase in the near future (Aristidou A., Pentila. M., Curr. Opin. Biotechnol, 2000, April, 11:2, 187-198). However, a great majority of published articles and patents, or patent applications, describe the utilization of cellulosic biomass by biocatalysts (bacteria and yeasts) for production of ethanol, which is expected to be useful as an alternative fuel. Such processes include fermentation of cellulosic biomass using different modified strains of Zymomonas mobilis (Deanda K. et al, Appl. Environ. Microbiol., 1996 December, 62:12, 4465-70; Mohagheghi A. et al, Appl. Biochem. Biotechnol., 2002, 98-100:885-98; Lawford H. G., Rousseau J. D., Appl. Biochem. Biotechnol, 2002, 98-100:429-48; PCT applications WO95/28476, WO98/50524), modified strains of Escherichia coli (Dien B. S. et al, Appl. Biochem. Biotechnol, 2000, 84-86:181-96; Nichols N. N. et al, Appl. Microbiol. Biotechnol., 2001 July, 56:1-2, 120-5; U.S. Pat. No. 5,000,000). Xylitol can be produced by fermentation of xylose from hemicellulosic sugars using Candida tropicalis (Walthers T. et al, Appl. Biochem. Biotechnol., 2001, 91-93:423-35). 1,2-propanediol can be produced by fermentation of arabinose, fructose, galactose, glucose, lactose, maltose, sucrose, xylose, and combination thereof using recombinant Escherichia coli strain (U.S. Pat. No. 6,303,352). Also, it has been shown that 3-dehydroshikimic acid can be obtained by fermentation of a glucose/xylose/arabinose mixture using Escherichia coli strain. The highest concentrations and yields of 3-dehydroshikimic acid were obtained when the glucose/xylose/arabinose mixture was used as the carbon source, as compared to when either xylose or glucose alone was used as a carbon source (Kai Li and J. W. Frost, Biotechnol. Prog., 1999, 15, 876-883). [0010] It is has been reported that Escherichia coli can utilize pentoses such as L-arabinose and D-xylose (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F. C. Neidhardt, ASM Press, Washington D.C., 1996). Transport of L-arabinose into the cell is performed by two inducible systems: (1) a low-affinity permease (K.sub.m about 0.1 mM) encoded by araE gene, and (2) a high-affinity (K.sub.m 1 to 3 .mu.M) system encoded by the araFG operon. The araF gene encodes a periplasmic binding protein (306 amino acids) with chemotactic receptor function, and the araG locus encodes an inner membrane protein. The sugar is metabolized by a set of enzymes encoded by the araBAD operon: an isomerase (encoded by the araA gene), which reversibly converts the aldose to L-ribulose; a kinase (encoded by the araB gene), which phosphorylates the ketose to L-ribulose 5-phosphate; and L-ribulose-5-phosphate-4-epimerase (encoded by the araD gene), which catalyzes the formation of D-xylose-5-phosphate (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F. C. Neidhardt, ASM Press, Washington D.C., 1996). [0011] Most strains of E. coli grow on D-xylose, but a mutation is necessary for the K-12 strain to grow on the compound. Utilization of this pentose is through an inducible and catabolite-repressible pathway involving transport across the cytoplasmic membrane by two inducible permeases (not active on D-ribose or D-arabinose), isomerization to D-xylulose, and ATP-dependent phosphorylation of the pentulose to yield D-xylulose 5-phosphate. The high-affinity (K.sub.m 0.3 to 3 .mu.M transport system depends on a periplasmic binding protein (37,000 Da) and is probably driven by a high-energy compound. The low-affinity (K.sub.m about 170 .mu.M) system is energized by a proton motive force. This D-xylose-proton-symport system is encoded by the xylE gene. The main gene cluster specifying D-xylose utilization is xylAB(RT). The xylA gene encodes the isomerase (54,000 Da) and xylB gene encodes the kinase (52,000 Da). The operon contains two transcriptional start points, with one of them being inserted upstream of the xylB open reading frame. Since the low-affinity permease is specified by the unlinked xylE, the xylT locus, also named as xylF (xylFGHR), probably codes for the high-affinity transport system and therefore should contain at least two genes (one for a periplasmic protein and one for an integral membrane protein) (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F. C. Neidhardt, ASM Press, Washington D.C., 1996). The xylFGH genes code for xylose ABC transporters, where xylF gene encodes the putative xylose binding protein, xylG gene encodes the putative ATP-binding protein, xylH gene encodes the putative membrane component, and xylR gene encodes the xylose transcriptional activator. [0012] Introduction of the above-mentioned E. coli genes which code for L-arabinose isomerase, L-ribulokinase, L-ribulose 5-phosphate 4-epimerase, xylose isomerase and xylulokinase, in addition to transaldolase and transketolase, allow a microbe, such as Zymomonas mobilis, to metabolize arabinose and xylose to ethanol (WO/9528476, WO98/50524). In contrast, Zymomonas genes which code for alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDH) are useful for ethanol production by Escherichia coli strains (Dien B. S. et al, Appl. Biochem. Biotechnol, 2000, 84-86:181-96; U.S. Pat. No. 5,000,000). [0013] A process for producing L-amino acids, such as L-isoleucine, L-histidine, L-threonine and L-tryptophan, by fermentation of a mixture of glucose and pentoses, such as arabinose and xylose, was disclosed earlier by authors of the present invention (Russian patent application 2003105269). [0014] However, at present, there are no reports describing bacteria having enhanced expression of the xylose utilization genes such as those at the xylABFGHR locus, or use of these genes for production of L-amino acids from a mixture of hexose and pentose sugars. SUMMARY OF THE INVENTION [0015] An object of present invention is to enhance production of an L-amino acid producing strain, to provide an L-amino acid producing bacterium having enhanced expression of xylose utilization genes, and to provide a method for producing L-amino acids from a mixture of hexose sugars, such as glucose, and pentose sugars, such as xylose or arabinose, using the bacterium. A fermentation feedstock obtained from cellulosic biomass may be used as a carbon source for the culture medium. This aim was achieved by finding that the xylABFGHR locus cloned on a low copy vector enhances production of L-amino acids, for example, L-histidine, L-threonine, L-lysine, L-glutamic acid and L-tryptophan. A microorganism is used which is capable of growth on the fermentation feedstock and is efficient in production of L-amino acids. The fermentation feedstock consists of xylose and arabinose along with glucose, as the carbon source. L-amino acid producing strains are exemplified by Escherichia coli strain. Thus the present invention has been completed. [0016] It is an object of the present invention to provide an L-amino acid producing bacterium of the Enterobacteriaceae family which has an enhanced activity of any of the xylose utilization enzymes. [0017] It is a further object of the present invention to provide the bacterium described above, wherein the bacterium belongs to the genus Escherichia. [0018] It is a further object of the present invention to provide the bacterium described above, wherein the bacterium belongs to the genus Pantoea. [0019] It is a further object of the present invention to provide the bacterium described above, wherein the activities of the xylose utilization enzymes are enhanced by increasing the expression amount of the xylABFGHR locus. [0020] It is a further object of the present invention to provide the bacterium described above, wherein the activities of the xylose utilization enzymes are increased by increasing the copy number of the xylABFGHR locus or modifying an expression control sequence of the genes so that the expression of the genes are enhanced. [0021] It is a further object of the present invention to provide the bacterium described above, wherein the copy number is increased by transforming the bacterium with a low copy vector harboring the xylABFGHR locus. Continue reading... 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